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www.aging-us.com 4688 AGING
www.aging-us.com AGING 2020, Vol. 12, No. 6
Priority Research Paper
ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging
Jing Zhao1,2,3,*, Lei Zhang1,2,*, Aiping Lu4,5, Yingchao Han7, Debora Colangelo1,6, Christina Bukata1, Alex Scibetta4,5, Matthew J. Yousefzadeh1,2, Xuesen Li1, Aditi U. Gurkar1, Sara J. McGowan1,2, Luise Angelini1,2, Ryan O’Kelly1,2, Hongshuai Li7, Lana Corbo1, Tokio Sano1, Heather Nick1, Enrico Pola6, Smitha P.S. Pilla8, Warren C. Ladiges8, Nam Vo7, Johnny Huard4,5, Laura J. Niedernhofer1,2, Paul D. Robbins1,2 1Department of Molecular Medicine and the Center on Aging, Scripps Research, Jupiter, FL 33458, USA 2Institute on the Biology of Aging and Metabolism and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55415, USA 3Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA 4Department of Orthopaedic Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030, USA 5Steadman Philippon Research Institute, Vail, CO 81657, USA 6Department of Orthopaedic Surgery, Catholic University of Rome School of Medicine, “A. Gemelli” University Hospital, Roma, Italy 7Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA 8Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA *Co-first author Correspondence to: Paul D. Robbins; email: [email protected] Keywords: ATM, NF-κB, DNA damage response, cellular senescence, aging Received: December 9, 2018 Accepted: Feburary 8, 2020 Published: March 22, 2020 Copyright: Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenitor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus, represents a therapeutic target to slow the progression of aging.
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INTRODUCTION
With aging there is an inevitable and progressive loss of
the ability of tissues to recover from stress, leading to the
increased incidence of chronic degenerative diseases. The
loss of tissue homeostasis is driven, in part, by an
increase in cellular senescence and a decline in stem cell
function, resulting in various aging-related diseases,
including osteoporosis, intervertebral disc degeneration,
chronic kidney disease, diabetes, neurodegeneration and
cancer [1–5]. Cellular senescence, characterized by
irreversible cell cycle arrest with sustained metabolic
activity, is a biological process that is physiologically
required during embryonic development, wound healing
and tumor suppression [6–10]. Importantly, senescent
cells can develop a senescence-associated secretory
phenotype (SASP), including expression of IL-6, IL-1α,
IL-1β and TNF-α, that affects neighboring cells, disrupts
stem cell niches, alters extracellular matrix, and induces
secondary senescence [8, 11, 12]. Cellular senescence is
mediated by p53/p21 and p16INK4a/retinoblastoma (Rb)
tumor suppressor pathways in response to stress [8, 13]
conferred by telomere attrition, DNA damage, oxidative
and inflammatory stress and oncogene dysregulation [8].
Cellular senescence directly contributes to the
progression of aging. For example, depletion of p16INK4a
positive cells in both progeroid and naturally aged
transgenic mice expressing an inducible apoptotic
transgene from the p16INK4a promoter leads to an
extension of healthspan [2, 3]. Similarly, clearance of
senescent cells using senolytic agents extends
healthspan, improves adult stem cell function and
extends lifespan in mice [4, 5, 14–17].
The DNA damage response (DDR), essential for
genome stability and organismal survival [18–21], is
mediated through pathways that recruit multi-protein
complexes to sites of DNA double-strand breaks
(DSBs) or stalled replication forks. In particular, the
MRE11-RAD50-NBS1 (MRN) complex is recruited to
sites of DSBs to facilitate the recruitment, retention and
activation of the ataxia-telangiectasia mutated (ATM)
kinase [22]. Autophosphorylation of ATM at Ser1981
enhances its kinase activity, leading to phosphorylation
of histone H2A variant H2AX (γH2AX) in nucleosomes
surrounding damaged sites and recruiting more ATM
and other repair factors [22, 23]. Additional DDR
proteins, including KRAB-associated protein-1 (KAP1),
p53 and checkpoint kinase 2 (CHK2), are
phosphorylated by ATM kinase, promoting DNA repair,
cell-cycle arrest, apoptosis and/or senescence [24, 25].
The Nuclear Factor κB (NF-κB) family of
transcription factors consists of five members in
mammalian cells, including RelA (p65), RelB, c-Rel,
p50/p105 and p52/p100 [26]. All NF-κB subunits
contain a Rel-homology domain (RHD), which is
essential for DNA binding activity and dimerization.
NF-κB functions to regulate innate and adaptive
immune responses, embryonic development,
proliferation, apoptosis, oncogenesis and senescence
[26]. The canonical NF-κB pathway is activated by
inflammatory stimuli, such as TNF-α, IL-1β and LPS,
which lead to the activation of the IκB kinase (IKK).
IKK is composed of two catalytic subunits, IKKα and
IKKβ, and one regulatory subunit, NF-κB essential
modifier (NEMO) or IKKγ [26]. Activated IKK
complex phosphorylates the inhibitory protein IκBα to
facilitate its polyubiquitination and degradation by the
26S proteasome, resulting in the translocation of the
NF-κB heterodimer into the nucleus where it regulates
gene transcription [26]. In addition, genotoxic stress
activates a TNF-α-independent, but ATM-dependent
NF-κB pathway via nuclear-localized NEMO [27, 28].
ATM phosphorylates NEMO at Ser85, which in turn
induces sumoylation and mono-ubiquitination of
NEMO at Lys277 and 309 [29]. These post-
translational modifications eventually lead to the
nuclear export of the ATM-NEMO complex to the
cytoplasm where it associates with ubiquitin and
SUMO-1 modified RIP1 and TAK1, activating the
catalytic IKKβ subunit [28, 30].
NF-κB activity increases in multiple tissues of humans
and rodents with aging and promotes cellular senescence
[31–36]. Genetic depletion of RelA/p65 in aged mouse
skin and a mouse model of human progeroid syndrome,
reversed gene expression signature of aging and aging
phenotypes [35, 37]. In addition, heterozygosity of
p65/RelA in a mouse model of Hutchinson-Gilford
Progeria Syndrome (Zmpste24-/-) resulted in attenuated
aging pathology and a prolonged lifespan, linked in part
to a reduced systemic inflammatory response and a
reduction in ATM/NEMO-mediated NF-κB activation
[34]. In addition, Nfkb1-/- (p50-/-) mice have increased
low-grade inflammation with signs of premature aging,
including neural degeneration, impaired regeneration and
declined overall lifespan [38–41]. Activation of NF-κB
also is associated with multiple aging-related chronic
diseases, including Alzheimer’s disease, Parkinson’s
disease, Type II diabetes, osteoporosis and
atherosclerosis [42], possibly through an increase in
secretion of SASP factors [43].
DNA damage is known to increase with aging as
demonstrated by an increase in DNA damage foci
(γH2AX) and oxidative DNA lesions (8,5’-
cyclopurines) [44, 45]. Intriguingly, persistent DDR
signaling mediated by ATM activation has been
reported to contribute to cellular senescence and SASP
[46]. In vitro, SASP is dependent on ATM activation,
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suggesting a molecular link between ATM and NF-κB
[8, 46, 47]. However, it is still unclear if aberrant DNA
damage-induced activation of ATM in vivo exacerbates
the cellular stress response to increase NF-κB,
senescence, SASP and subsequently aging.
To address the role of ATM in driving NF-κB mediated
senescence and aging, we used Ercc1-/Δ mice that model a
human progeroid syndrome caused by impaired repair of
DNA damage. The mice express only 5% of the normal
level of the DNA repair endonuclease ERCC1-XPF that is
required for nucleotide excision, interstrand crosslink and
repair of some double-strand breaks. As a consequence,
the Ercc1-/Δ mice spontaneously and rapidly develop
progressive age-related diseases, including osteoporosis,
sarcopenia, intervertebral disc degeneration,
glomerulonephropathy, neurodegeneration, peripheral
neuropathy and loss of cognition [48].
Here, we demonstrate that ATM and downstream
effectors are persistently elevated in Ercc1-/∆ and
naturally aged mice, concomitant with hyperactive NF-
κB signaling. Reducing ATM activity either genetically
or pharmacologically reduced cellular senescence and
downregulated NF-κB activation in cell culture.
Importantly, Ercc1-/Δ mice heterozygous for Atm
exhibited significantly reduced NF-κΒ activity, reduced
cellular senescence, improved muscle-derived
stem/progenitor cell function and attenuated age-related
bone and intervertebral disc pathologies, leading to an
extension of healthspan. Similarly, inhibiting ATM in
Ercc1-/∆ mice by treatment with the ATM inhibitor KU-
55933 reduced senescence and SASP marker expression.
These results demonstrate a key role for ATM in aging
and suggest that it is a therapeutic target for delaying or
improving numerous age-related diseases.
RESULTS
NF-κB and ATM signaling are highly activated in
cellular senescence, as well as accelerated and
natural aging
Our previous studies using transgenic mice carrying a
NF-κB-dependent EGFP reporter demonstrated an
increase in the percentage of EGFP-positive cells in the
liver, kidney, skeletal muscle and pancreas of progeroid
Ercc1-/Δ and aged wild-type (WT) mice [35]. To further
quantify NF-κB activation with aging, p-p65 (Ser536), a
marker of NF-κB activation, was measured in murine
liver (Figure 1A). Phosphorylation of p65 was
significantly increased in 16-week-old Ercc1-/Δ mice
compared to age-matched WT mice (Supplementary
Figure 1A). In addition, there was an increase in the
level of p-ATM as well as two senescence markers,
γH2AX [49] and p21, in Ercc1-/∆ liver compared to WT
controls (Figure 1B and Supplementary Figure 1B). To
determine if NF-κB and ATM were activated in WT
mice with aging, p-p65, p-IκBα and p-ATM were
measured by immunoblot in liver extracts from WT
mice at multiple ages. The levels of p-p65 and p-IκBα
increased gradually with age from 3 to 12 and 24-
months of age (Figure 1C and Supplementary Figure
1C). These correlated with increased levels of p-ATM
and the senescence marker p21 at 12 and 24 months of
age (Figure 1D and Supplementary Figure 1D).
To examine ATM and NF-κΒ activation with
senescence, the phosphorylation of ATM and NF-κΒ
targets were measured initially in primary Ercc1-/-
mouse embryonic fibroblasts (MEFs), which undergo
premature senescence at 20% O2. The levels of p-ATM
and p-KAP1 were increased in Ercc1-/- MEFs compared
to WT MEFs (Figure 1E). Moreover, p-p65 levels were
increased in passage 5 Ercc1-/- MEFs compared to WT
cells (Figure 1F). There also was an increase in nuclear
staining of p65 and NEMO in Ercc1-/- MEFs compared
to WT cells, indicating NF-κB activation through a
NEMO-dependent manner (Figure 1G). These findings
suggest that NF-κB and ATM are co-activated in cells
and tissues with higher levels of DNA damage-induced
senescence.
Pharmacologic inhibition of ATM rescues
senescence caused by genotoxic stress via
suppressing NEMO-dependent NF-κB activation
To elucidate further the causative role of activated ATM
and DDR signaling in driving senescence, the effect of a
selective ATM kinase inhibitor, KU-55933 [50], on
senescence in MEFs was examined. Treatment of DNA
repair deficient Ercc1-/- MEFs with KU-55933 (10 μM)
reduced the percent of SA-βgal positive cells to a level
similar to WT MEFs (Figure 2A and 2B). Additional
markers of senescence, including the cell-cycle regulators
p21Cip1 and p16INK4A, also were decreased by KU-55933
treatment (Figure 2C). As expected, autophosphorylation
of ATM at Ser1981 was downregulated by the ATM
inhibitor, as were the levels of p-KAP1 and γH2AX
(Figure 2D). Interestingly, ATM inhibition also
decreased Poly [ADP-ribose] polymerase 1 (PARP1)
abundance (Figure 2D), an enzyme that promotes DNA
repair and chromatin remodeling, utilizing NAD+ as a
cofactor [51]. Interestingly, our results also suggest that
inhibition of ATM activity may regulate ATM expression
at protein level as indicated by reduced ATM level
(Figure 2D). Furthermore, ATM inhibition reduced the
abundance of nuclear-localized p65 and NEMO and the
level of p-p65 (Figure 2E), as well as NF-κB
transcriptional activity, measured using a NF-κB
luciferase reporter assay (Figure 2F). Finally, treatment
with the ATM inhibitor significantly reduced expression
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of multiple senescence and SASP markers as determined
by qRT-PCR (Figure 2G). Taken together, these results
suggest that ATM activation triggered by endogenous
DNA damage plays a critical role in driving cellular
senescence, SASP and NF-κB activation in a NEMO-
dependent manner.
Genetic depletion of Atm decreases genotoxic stress-
induced cellular senescence
To eliminate possible off-target effects of the ATM
inhibitor KU-55933, Ercc1-/- MEFs heterozygous for
Atm (Ercc1-/-Atm+/-) were generated. The Ercc1-/-Atm+/-
Figure 1. DDR and NF-κB are activated concomitantly in senescent MEFs and aged tissues. (A) Immunoblot detection of p-p65
and total p65 in liver tissue from 16-week-old WT (n=3) and Ercc1-/Δ (n=3) mice. (B) Immunoblot detection of phosphorylation of ATM and downstream targets γH2AX and p21 in liver from 16-week-old WT and Ercc1-/Δ mice. (C) Immunoblot detection of phosphorylation of NF-κB and IκBα in liver lysates from 3, 12 and 24 month-old WT mice. n=3 mice per group. (D) Immunoblot detection of p-ATM, ATM and p21 in the same liver lysates. (E) Immunoblot detection of DDR effectors in nuclear extracts from passage 5 WT and Ercc1-/- MEFs, grown at 20% oxygen. (F) Level of NF-κB activation is higher in Ercc1-/- MEFs compared to WT MEFs at passage 5, as measured by Immunoblot detection of p-p65 and total p65 in WT and Ercc1-/- MEFs at passage 5 after culturing in 20% oxygen. (G) Representative images of immunofluorescent detection of p65 and NEMO in passage 4 WT and Ercc1-/- MEFs grown at 20% oxygen. Blue: DAPI staining; Green: p65 (top panel) or NEMO (bottom panel). Images were taken at the magnification of 60x.
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Figure 2. Pharmacologic inhibition of ATM rescues oxidative stress-induced senescence by suppressing ATM- and NEMO-mediated NF-κB activation. (A) Representative images of primary WT and Ercc1-/- MEFs were induced to undergo senescence by serial
passaging at 20% oxygen. At passage 5, MEFs were grown in the presence or absence of KU-55933 (10 μM) for 72 hrs. Senescence was determined by SA-βgal staining. Images were obtained at the magnification of 10x. (B) Quantitation of the percent SA-βgal positive cells. Graph represents the mean +/- s.e.m. of three independent experiments. Student’s t-test, ***p <0.001, ****p <0.0001. (C) Passage 5 Ercc1-/- MEFs treated with vehicle or KU-55933 (10 μM) for 72 hours were collected and levels of p21 and p16INK4a were determined by western blotting. (D) Passage 5 Ercc1-/- MEFs were treated with KU-55933 (10 μM) for 72 hours and whole cell lysate (CL) and nuclear extracts (NE) were analyzed by immunoblotting for expression of proteins involved in the DNA damage response. (E) Whole cell lysate (CL) and nuclear
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extract (NE) were extracted from Ercc1-/- MEFs treated with 10 μM of KU-55933 for analysis of nuclear NEMO and p65. GAPDH was used as a loading control of total proteins and LaminA/C as a loading control of nuclear protein. (F) Passage 5 WT and Ercc1-/- MEFs transfected with a NF-κB-luciferase reporter construct were cultured in the presence or absence of KU-55933 (10 μM) and were collected for luciferase assays after 72 hours. (G) qRT-PCR analysis of mRNA expression in passage 5 WT and Ercc1-/- MEFs treated with or without of KU-55933 (10 μM) for 72 hrs. P values were determined using a Student’s t-test. *p<0.05, **p<0.01, ***p <0.001.
MEFs had increased proliferation compared to Ercc1-/-
MEFs (Figure 3A). There also was a reduction in the
percent of SA-ßgal+ Ercc1-/-Atm+/- MEFs compared to
Ercc1-/- MEFs (Figure 3B and 3C). Expression of p16INK4a
also was reduced in the double mutant MEFs (Figure 3D).
Finally, there was a reduction in the level of secreted IL-6,
a SASP factor, in conditioned media from Ercc1-/-Atm+/-
MEFs compared to Ercc1-/- cells (Figure 3E). Taken
together, these results suggest that ATM promotes
genotoxic stress-induced cellular senescence, in part,
through the activation NF-κB signaling.
Genetic depletion of Atm extends healthspan in
Ercc1-/Δ mice by reducing cellular senescence
To determine if Atm heterozygosity extends healthspan
in Ercc1-/∆ mice, age-related symptoms, including
kyphosis, tremor, ataxia, gait disorder, hind limb muscle
wasting, forelimb grip strength and, in particular,
dystonia were measured weekly in Ercc1-/∆ and Ercc1-
/∆Atm+/- mice. As shown in Figure 4A and 4B, Atm
heterozygosity reduced the severity and slowed
progression of aging symptoms in Ercc1-/Δ mice.
To determine if the extended healthspan correlated with
reduced cellular senescence in Ercc1-/∆Atm+/- mice, the
levels of expression of senescent markers and SASP
factors were measured by qRT-PCR analysis.
Expression of p21 was significantly reduced in 12-week-
old Ercc1-/∆Atm+/- livers compared to those from aged-
matched Ercc1-/∆mice, as was IL-6, a SASP factor and
NF-κB target gene (Figure 4C). Similarly, the expression
of senescence markers and SASP factors (except p16)
were significantly reduced in Ercc1-/∆Atm+/-
Figure 3. Oxidative stress-induced cellular senescence is reduced by genetic depletion of Atm. (A) Proliferation of WT (black),
Ercc1-/- (blue) and Ercc1-/-Atm+/- (red) MEFs serially passaged at 20% oxygen was measured by an automated cell counter. Data shown are representative of three independent experiments using distinct MEF lines. (B) SA-βgal staining of serially passaged MEFs cultured at 20% oxygen. Shown are representative images of passage 5 WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs taken at 10x magnification. (C) The average percentage of SA-βgal positive cells at each indicated passage. Ten fields were acquired and quantified per sample. Data shown are representative of two independent experiments. (D) Senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs (passage 5) cultured at 20% oxygen for 72 hrs were collected and lysed for immunoblot analysis of p16INK4a. (E) Supernatant collected from senescent WT, Ercc1-/- and Ercc1-/-Atm+/- MEFs was analyzed by ELISA for secreted IL-6. Graphs represent mean+/- s.e.m. P value was determined using Student’s t-test. *p<0.05, **p<0.01.
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quadriceps compared with those in Ercc1-/∆mice (Figure
4D). In wildtype mice, there was no effect of ATM
heterozygosity on expression of these senescence and
SASP markers. Interestingly, IL-6 and TNF-α, but not
p21Cip1 or p16INK4a expression, were significantly lower
in liver of 12-week-old Ercc1-/Δ p65+/- mice compared
to age-matched Ercc1-/ΔAtm+/- mice (Figure 4E). This
indicates a distinct role for NF-κB in promoting aging.
Taken together, these results suggest that genetic
reduction of ATM leads to a reduction in cellular
senescence and aging symptoms in vivo.
To determine if pharmacologic inhibition of ATM in
Ercc1-/∆ mice confers a similar reduction in senescence
and SASP markers as ATM heterozygosity, Ercc1-/∆
mice were treated three times per week i.p. for 2 weeks
with 10 mg/kg of KU-55933. The mice were then
analyzed for the extent of senescence and SASP in
different tissues. As shown in Figure 4F, the levels of
expression of senescent markers and SASP factors were
measured were significantly reduced in liver tissues
from Ercc1-/∆ mice compared to untreated controls.
Atm haploinsufficiency improves function of muscle-
derived stem/progenitor cells (MDSPC)
A decline in stem cell function has long been associated
with aging, which in the musculoskeletal system leads
to sarcopenia and muscle wasting [1, 52]. We
previously reported that muscle-derived stem/progenitor
cells (MDSPCs) isolated from Ercc1-/Δ mice failed to
proliferate or differentiate properly, similar to MDSCPs
from naturally aged mice [53]. Interestingly, the
function of MDSPCs isolated from Ercc1-/∆ mice was
partially restored by Atm heterozygosity (Figure 5). In
fact, MDSPCs isolated from Ercc1-/∆Atm+/- mice
showed similar levels of myogenic differentiation to
WT mice (Figure 5A, 5C and 5D). Proliferation of
MDSPCs isolated from Ercc1-/∆Atm+/- mice also
increased by 50% compared to control MDSPCs
(Figure 5B). This result is consistent with delayed onset
and reduced severity of gait disorder observed in Ercc1-
/ΔAtm+/- mice, which is partly due to severe muscle
wasting (data not shown).
Atm haploinsufficiency improves aging-related
pathology in certain tissues
To further assess the effect of ATM status on aging,
tissues from 12-week Ercc1-/∆ and Ercc1-/∆Atm+/- mice
were examined histologically. There are reduced
lymphoid aggregates in the liver and kidney of Ercc1-/∆
and Ercc1-/∆Atm+/- mice, suggesting reduced
inflammatory infiltration in tissues at the age analyzed
[54]. In addition, Ercc1-/∆Atm+/- mice had reduced age-
related bony changes in the lumbar vertebrae, as
determined using μCT. Compared to WT mice, Ercc1−/∆
mice showed marked trabecular bone loss (Figure 6A),
signified by an increase in osteoporosis (1-BV/TV), a
decrease in trabecular number (Tb.N) and trabecular
thickness (Tb.Th) and an increase in the trabecular
spacing (Tb.Sp). Atm heterozygosity significantly
improved bone qualities when compared with Ercc1−/∆
mice, showing reduced vertebral osteoporosis and
trabecular spacing, accompanied by a significant
increase in trabecular number (Figure 6A). However, no
significant difference was found in trabecular thickness
(Figure 6A). Ercc1-/∆Atm+/- mice also had improved
Safranin O staining of the intervertebral disc from the
lumbar spines, consistent with significantly higher
levels of glycosaminoglycans (GAGs) in the nucleus
pulposus (Figure 6B and 6C) [55–57]. GAG
measurement indicates the level of the matrix
proteoglycans, which plays a critical role in
counteracting mechanical forces imparted on the spine
[58]. Taken together, these results suggest that
heterozygosity of Atm improves certain age-related
conditions, in particular in the musculoskeletal system.
Atm haploinsufficiency reduces NF-κB signaling in
Ercc1-/Δ mice
To determine the extent to which ATM signaling
mediates NF-κB activation in vivo, livers from 12- and
16-week-old Ercc1-/Δ and Ercc1-/∆Atm+/- mice were
examined. There was a significant decrease in DDR
signaling in Ercc1-/∆Atm+/- mice compared to Ercc1-/Δ
mice as indicated by reduced levels of p-ATM and
γH2AX (Figure 7A, 7C and Supplementary Figure 2A,
2C). Moreover, the levels of p21Cip1 were significantly
decreased in livers from Ercc1-/∆Atm+/- mice,
particularly at 12 weeks of age, consistent with reduced
cellular senescence and the qRT-PCR results (Figure
4C). Importantly, the level of phospho-p65 was greatly
reduced in Ercc1-/∆Atm+/- livers (Figure 7B, 7D and
Supplementary Figure 2B, 2D), suggesting that NF-κB
activation in Ercc1-/Δ mice is driven, at least in part, by
ATM activation. Accordingly, there was reduced p-
IκBα and IκBα levels in Ercc1-/∆Atm+/- mice compared
to Ercc1-/Δ controls (Figure 7B, 7D and Supplementary
Figure 2B, 2D) [59, 60].
To determine if the molecular changes in Ercc1-/∆Atm+/-
mice mirror that in Ercc1-/Δp65+/- mice, ATM and NF-
κB activation was measured in Ercc1-/Δ and Ercc1-
/Δp65+/- mice. There was no difference in the level of
ATM or p-ATM between Ercc1-/Δp65+/- and Ercc1-/Δ
mice (Figure 7E and Supplementary Figure 2E),
suggesting that DDR/ATM signaling is the upstream
regulator of NF-κB. The levels of p-p65 and total IκBα
were reduced in Ercc1-/Δp65+/- mice compared to Ercc1-
/Δ mice (Figure 7F and Supplementary Figure 2F),
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Figure 4. Genetic reduction of Atm attenuates aging phenotypes and reduces cellular senescence in vivo. (A) Representative
images (left panel) of 15-week-old Ercc1-/Δ and Ercc1-/ΔAtm+/- mice illustrating the severity of their dystonia. (B) The composite score of aging symptoms (right panel) was plotted at the indicated ages. n=8-10 mice per group. (C) qRT-PCR analysis of mRNA expression in liver from 12-week-old WT, Atm+/-, Ercc1-/Δ and Ercc1-/ΔAtm+/- mice. n=3-6 per group. (D) qRT-PCR analysis of mRNA expression in quadriceps from 12-
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week-old WT, Atm+/-, Ercc1-/Δ and Ercc1-/ΔAtm+/- mice. n=3-11 per group. (E) qRT-PCR analysis of mRNA expression in liver from 10 to 12-week-old WT, p65+/-, Ercc1-/Δ and Ercc1-/Δp65+/- mice. n=4-5 per group. (F) mRNA expression of senescence markers in the liver of 12-week-old Ercc1-/Δ mice treated with 10 mg/kg of KU-55933 intraperitoneally 3 times per week for two weeks. n = 3 per group. Graphs represent mean+/- s.e.m. P value was determined using Student’s t-test. *p<0.05, **p<0.01, ***p <0.001, ****p <0.0001.
consistent with qRT-PCR results (Figure 4E) showing
reduced NF-κB-mediated transcription. Taken together,
these results suggest that DDR signaling plays an
essential role in NF-κB activation in response to
endogenous DNA damage. Moreover, the deletion of
one Atm allele is sufficient to attenuate DDR signaling
and dampen NF-κB activation in vivo.
DISCUSSION
DNA damage is a critical factor in driving cellular
senescence and aging [61]. Multiple human diseases of
accelerated aging, such as XFE progeroid syndrome,
Cockayne syndrome, Hutchinson-Gilford progeria and
Werner syndrome are caused by inherited defects in
maintaining genome integrity [48, 62]. Levels of
oxidative DNA damage are increased in old or progeroid
organisms compared to young [44, 63, 64]. Although the
ATM kinase is a critical mediator of the DNA damage
response, in vivo evidence linking chronic activation of
ATM to senescence and aging is still lacking. Here, we
demonstrate that ATM activation increases with aging in
mammals. In addition, in the Ercc1-/∆ progeroid mouse
model of accelerated aging, genetic reduction of ATM
reduces cellular senescence, improves stem cell function,
extends healthspan and reduces certain age-related
pathologies in the musculoskeletal system. Moreover, we
demonstrate that ATM promotes senescence and aging, at
least in part, by regulating the transcriptional activity of
NF-κB (Figure 8).
ATM activity is increased in liver with aging in not only
progeroid Ercc1-/Δ mice, but also naturally aged WT
mice. Similarly, there was an increase in NF-κB activity
Figure 5. Atm heterozygosity improves muscle stem cell function and muscle regeneration. Myogenic progenitor cells (myoblasts)
and MDSPCs were isolated via preplate technique, 3 days after myoblasts were obtained, and the bright field pictures were taken. A total of three populations of Atm+/-, Ercc1-/∆Atm+/- and Ercc1-/∆ were isolated from distinct mice and tested. All scale bars = 100 µm. (A) MDSPCs were cultured in myogenic differentiation medium for 3 days. Bright field images were taken and the cell fusion into multinucleated myotubes was determined by immuno-staining for MyHCf, a terminal myogenic differentiation marker. (B) Cell proliferation of MDSPCs was measured using an MTS assay. The graph displays the average of three populations. Error bars indicate mean ± SD. ***P<0.001 (C) Representative images of immunofluorescence detection of differentiated myofibers. All scale bars in panel C=50 μm. (D) Myogenic differentiation was quantified by determining the number of nuclei in MyHCf positive myotubes relative to the total number of nuclei in the culture. Error bars indicate “mean ±SD”. *P<0.05. **P<0.01. Error bars indicate “mean ± SD”. *P<0.05. Two-tailed Student’s t-test was performed.
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in livers that correlates with ATM activity. Conversely,
a reduction in ATM activation in vivo either genetically
or pharmacologically resulted in a reduction in the
levels of γH2AX in liver and decreased expression of
senescent markers and SASP, in particular p21Cip1 and
Il6 (Figure 4C and 7A, 7C). Consistent with these
observations, there was an increase in ATM and NF-κB
activity in ERCC1-deficient cells grown under oxidative
stress conditions in cell culture. Reduction of ATM
either genetically or pharmacologically in MEFs also
resulted in a reduction in oxidative stress-induced
senescence along with reduction in NF-κB activation.
We previously demonstrated that heterozygosity in
p65/RelA (Ercc1-/Δp65+/-) in Ercc1-/Δ mice resulted in
reduced senescence in multiple tissues as well as
extended healthspan. Here we demonstrate that Atm
heterozygosity reduced NF-κB activation to an extent
similar to p65 heterozygosity in Ercc1-/Δ mice,
suggesting that ATM kinase is a major activator of NF-
κB in the context of DNA-damage mediated senescence
and aging. As a result, expression of SASP factors
transcriptionally regulated by NF-κB, especially IL-6,
was down-regulated in livers of both Ercc1-/Δp65+/- and
Ercc1-/∆Atm+/- mice. These findings support previous
studies reporting that ATM activation is indispensable
for the SASP phenotype secreting inflammatory
cytokines [46, 65]. Interestingly, p65/RelA
heterozygosity resulted in a stronger reduction in IL-6
and TNF-α expression compared to Atm heterozygosity,
suggesting either an ATM-independent pathway or that
heterozygosity of Atm has less of an effect on the
pathway. We speculate that DSBs activate NF-κB
primarily through an ATM/NEMO-dependent pathway,
Figure 6. Genetic reduction of Atm improves bone and intervertebral disc pathology in progeroid Ercc1-/Δ mice. (A)
Representative micro-CT images of lumber spines comparing severity of osteoporosis in 16-week-old WT, Ercc1-/Δ, Ercc1-/ΔAtm+/- mice. n=3-5 per group. Quantification of vertebral porosity, trabecular number, trabecular separation, thickness of trabecular bone was performed and shown. (B) Safranin O staining for disc matrix in thoracic discs from 12-week-old Ercc1-/Δ and Ercc1-/ΔAtm+/- mice. (C) GAG content measured by DMMB assays with NP tissues isolated from 12-week-old lumber discs. n=3 each group. Mean+/- s.e.m. P value was determined using Student’s t-test. **p<0.01.
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which then increases the production of TNF-α and other
SASP factors. These SASP factors, in turn, trigger a
second wave of NF-κB activation, enhancing cell-
autonomous and cell-non-autonomous senescence
[30, 66] (Figure 8). Thus, Atm haploinsufficiency only
dampens the primary activation of NF-κΒ, while p65
heterozygosity targets both primary and secondary NF-
κB activation, conferring a stronger inhibition of the
SASP phenotype. These results also are consistent with
a greater effect of heterozygosity in p65/RelA on liver
and kidney pathology than Atm heterozygosity.
We previously reported that reduction in p65/RelA in
muscle derived stem/progenitor cells (MDSPCs) from
Ercc1-/∆ mice improved their ability to proliferate as
well as differentiate. Here we demonstrated that
reduction in ATM activity also improved self-renewal
and differentiation in muscle derived stem/progenitor
cells, suggesting persistent activation of ATM-
dependent signaling negatively regulates stem cell
function (Figure 5). This consistent with the previous
observation that depletion of p21Cip1, which is regulated
by ATM-p53, restores stem cell self-renewal and tissue
homeostasis without accelerating carcinogenesis in
mice deficient in telomerase [67].
Moreover, we and others previously demonstrated that
genetic and pharmacological reduction in p65/RelA
improves bone architecture and reduces osteoporosis [34,
35]. Here we demonstrated that Atm haploinsufficiency
also leads to significantly improved osteoporosis and
reduced disc degeneration (Figure 6), suggesting ATM
activation could contribute to increased chronic
inflammation in spines via activating NF-κB in aging.
Figure 7. ATM and NF-κB activation are downregulated in Ercc1-/Δ mice heterozygous for Atm. (A) Livers were collected at 12
weeks of age from WT, Ercc1-/Δ and Ercc1-/ΔAtm+/- mic (n=3 per genotype) and lysates analyzed by western blot for activation of ATM and its downstream effectors. (B) Same liver lysates were used to measure phosphorylation of p65 and IκBα. (C) Western blot analysis of livers from 16-week-old WT, Ercc1-/Δ and Ercc1-/ΔAtm+/-mice (n=3 per genotype) probed for activation of ATM. GAPDH was used as a loading control. (D) Same liver lysates used to measure activation of NF-κB. (E) Fourteen-week-old livers from Ercc1-/Δ and Ercc1-/Δp65+/- mice (n=3 per genotype) were analyzed by western blot for activation of ATM (F) and NF-κB.
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Persistent DNA damage-mediated DDR signaling, in
particular ATM activation, has been demonstrated to be
essential for the establishment of an NF-κB-dependent
SASP phenotype in cell culture. Nuclear translocation of
NEMO plays a critical role in relaying nuclear signal to
cytoplasm to activate NF-κB in response to genotoxic
stress [28]. For example, increased ATM
phosphorylation and nuclear-localized NEMO were
found in the Zmpste24-/- mouse model of Hutchinson-
Gilford progeria syndrome (HGPS), where accumulation
of nuclear prelamin-A leads to a perturbation of NF-κB
signaling [34]. The transcription factor GATA4 was
identified as a key mediator connecting DDR signaling to
NF-κB activation and the senescent SASP phenotype
[13]. In addition, ATM/IFI16 mediated non-canonical
activation of DNA sensing adaptor STING was
demonstrated recently in etoposide-induced nuclear DNA
damage, leading to increased NF-κB activation [68].
However, in this study, we observed increased nuclear
localization of NEMO in response to nuclear DNA
damage, which could be ablated by an ATM inhibitor.
Thus, our results support the presence of ATM/NEMO-
dependent NF-κB pathway in response to chronic DNA
damage.
ATM kinase is best known for its causal role in ataxia
telangiectasia (AT), a rare autosomal recessive disease
characterized by progressive neurodegeneration,
immunodeficiency, cancer predisposition,
radiosensitivity and premature skin aging [69–71].
Despite the fact that Atm-null mice have reduced
dopaminergic neurons with age, decreased synaptic
function in hippocampal neurons and defects in neuronal
network activity, mice heterozygous for Atm improved
not only neurodegenerative pathology, but also
Huntington-like behavior in a mouse model of
Huntington’s disease [22, 72, 73]. In addition, genetic
and/or pharmacologic reduction of ATM reduced
doxorubicin-induced cardiotoxicity and rescued cardiac
inflammation and heart failure caused by DNA single-
strand breaks [74, 75]. These observations are consistent
with our results showing neurologic symptoms and
musculoskeletal pathology were improved in Atm
haploinsufficient Ercc1-/∆ mice. In addition, our study
showed that a short-term administration of KU55933
reduced multiple senescence and SASP markers in liver
tissues in Ercc1-/∆ mice. These results indicate that ATM
kinase may be a potential drug target for the treatment
of multiple aging-related diseases, especially those
Figure 8. A model depicting how endogenous nuclear DNA damage activates NF-κB via an ATM- and NEMO-dependent mechanism to drive cellular senescence and senescence-associated secretory phenotype (SASP). In response to chronic
accumulation of endogenous DNA damage, ATM undergoes autophosphorylation and promotes phosphorylation, SUMOylation, and monoubiquitylation of NEMO. As a result, monoubiquitylated NEMO along with ATM translocates to the cytoplasm, activating the IKK complex. Phosphorylation of IκB leads to the release of p65 so that it can translocate into nucleus upregulating a transcriptional program of certain SASP factors, such as TNFα and IL-6. Secreted SASP factors then trigger a second wave of NF-κB activation through cytokine receptors, further enhancing cell-autonomous pathway-mediated senescence and inducing non-cell-autonomous pathway-mediated senescence.
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that have strong correlation with DNA damage
accumulation. Given that heterozygous carriers of AT are
symptom-free in general, we speculate that one WT allele
of Atm is sufficient to exert its function in DNA repair
and thereby doesn’t trigger the neurological degeneration
as seen in AT patients. Interestingly, studies in Chinese
and Italian nonagenarians/centenarians have identified a
single nucleotide polymorphism (SNP, rs189037) of in
the promoter region of ATM that moderately represses
transcription of Atm by regulating its binding to an
activator protein 2α (AP-2α). These results suggest that,
similar to our results in Ercc1-/∆ mice, a slight reduction
in ATM can contribute to extended lifespan in humans
[76, 77].
Taken together, our results suggest that ATM acts as the
main stimulus of NF-κB activation in DNA damaged-
induced senescence and aging. Reduction in ATM
either genetically or pharmacologically is able to reduce
the adverse effects of chronic DNA damage, reducing
cellular senescence, improving stem cell function and
extending healthpsan. Thus, ATM and NF-κB represent
therapeutic targets, at least later in life, for improving
frailty and certain aging-related diseases.
MATERIALS AND METHODS
Cells and mice
Primary mouse embryonic fibroblasts (MEFs) were
isolated on embryonic day 12.5-13.5. In brief, mouse
embryos were isolated from yolk sac followed by
removal of viscera, lung and heart. Embryos were then
minced into fine chunks, covered with media, cultured
at 3% oxygen to reduce stresses and serially passaged.
MEFs were grown in 1:1 of Dulbecco’s Modification of
Eagles Medium (with 4.5 g/L glucose and L-glutamine)
and Ham’s F10 medium, supplemented with 10% fetal
bovine serum, penicillin and streptomycin and non-
essential amino acids. To induce oxidative stress and
oxidative DNA damage, MEFs were switched to 20%
oxygen at passage 3.
Ercc1+/- and Ercc1+/Δ mice from C57BL/6J and FVB/NJ
backgrounds were crossed to generate Ercc1-/Δ F1
hybrid mice. Atm+/- mice were crossed to Ercc1+/- from
C57BL/6J background to generate Ercc1+/-Atm+/- mice,
which were then bred with Ercc1+/Δ mice from FVB/NJ
background to generate F1 Ercc1-/∆Atm+/- mice.
Breeders were backcrossed for ten generations yielding
F1 mice that are genetically identical. Animal protocols
used in this study were approved by Scripps Florida
Institutional Animal Care and Use Committee.
12-week-old Ercc1-/Δ mice were injected
intraperitoneally with 10 mg/kg of KU-55933 (AdooQ
Bioscience, Irvine, CA, USA) or the same volume of
vehicle (1% DMSO: 30% PEG400: 69% H2O) 3 times
per week for two weeks. All mice were euthanized 1
hour after the last injection.
Immunoblotting
MEFs were treated with either KU-55933 (10 μM,
Selleckchem) or vehicle for 72 hours prior to harvesting
for analysis of protein expression. Cell lysates were
prepared with RIPA buffer (20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM Na2EDTA, 1 μM EGTA, 1% NP-
40, 1% sodium deoxycholate, 2.5 μM sodium
pyrophosphate, 1 μM β-glycerophosphate, 1 μM
Na3VO4 and 1 µg/ml leupeptin), supplemented with 1X
of protease inhibitor cocktails (Sigma) and Halt
phosphatase inhibitor cocktail (Thermo #78420). Snap-
frozen liver and kidney samples were homogenized
using FastPrep-24 instrument in RIPA buffer. Equal
amounts of proteins (40 μg) were resolved on 4-15%
Mini-PROTEAN TGX precast protein gels (Bio-Rad).
Primary antibodies used are as follows: p-ATM Ser
1981 (cell lysates: Rockland cat. no. 200-301-400;
tissue lysates: Santa Cruz sc-47739), ATM (CST
#2873), p-KAP1 Ser 824(Abcam ab70369), KAP1
(Abcam ab22553), γ-H2AX (Novus NB100-384),
PARP1 (CST #9532), p-p65 Ser 536 (CST #3033), p65
(CST #8242), p-IκBα Ser 32/36 (CST #9246), IκBα
(Santa Cruz sc-371), p16INK4a (Santa Cruz sc-1207),
p21Cip1 (Santa Cruz sc-6246), LaminA/C (Santa Cruz
sc-20681), anti-tubulin (CST #2146), and GAPDH
(CST #5174). Blots were exposed to X-ray film in a
dark room or developed on iBright™ FL1000 Imaging
System (Figure 1E and 1F). Protein levels were
quantified by Image J software.
Nuclear extraction
Extraction of cytoplasmic and nuclear fractions was
performed using the NE-PER nuclear and cytoplasmic
extraction reagents (Thermo Fisher) according to the
manufacturer’s instructions. Briefly, 1x106 cells were
suspended and lysed in CERI and CERII reagents
consecutively on ice to obtain cytoplasmic fractions.
Pellets of intact nuclei were then suspended in NER
reagent to release nuclear contents.
NF-κB luciferase reporter assay
Primary MEFs transfected with a NF-κB luciferase
reporter construct were cultured in 6-well plates in
triplicate in the absence or presence of KU-55933 (10
μM) for 72 hrs. Cells were then collected with Passive
Lysis Buffer (Promega) and luciferase assay (Promega)
was performed by using a luminometer according to the
manufacturer’s instructions.
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Cell proliferation assay
Passage 3 MEFs were seeded at 5x105 cells in 10-cm
plates, allowed to grow for 72-96 hours to reach
confluence at 20% oxygen and then trypsinized for
determination of cell number. Serial passage was
carried until passage 5 and cell number was determined
for each passage. Cell number was measured using a
Moxi Z Mini automated cell counter. Log cell number
was plotted versus passage number.
Enzyme-linked immunosorbent assay (ELISA)
Supernatant collected at the end of passage 4 from
primary MEFs was analyzed for IL-6 production by
ELISA using a mouse IL-6 ELISA kit (Becton
Dickinson) according to the manufacturer’s instructions.
Quantitative reverse transcription- polymerase
chain reaction (qRT-PCR)
Snap-frozen tissues were preserved in RNAlater
stabilization solution (Thermo Fisher). Total RNA was
extracted using TRIZOL reagent (Life Technologies)
and 1.5 μg of RNA was subjected to complementary
DNA (cDNA) synthesis using SuperScript VILO cDNA
synthesis kit (Thermo Fisher). qRT-PCR was performed
with Platinum SYBR Green qPCR SuperMix-UDG with
ROX (Thermo Fisher) in a StepOnePlus Real-Time
PCR system. Relative expression of target genes was
calculated using the comparative CT method (ΔΔCT).
ΔCT was calculated by normalizing to an internal
control gene Actb (β-actin) and ΔΔCT by normalizing to
the mean ΔCT value of the control group. Primers used
are as follows: Cdkn1a (p21Cip1) forward:
GTCAGGCTGGTCTGCCTCCG; Cdkn1a (p21Cip1)
reverse: CGGTCCCGTGGACAGTGAGCAG; Cdkn2a
(p16INK4a) forward: CCCAACGCCCCGAACT; Cdkn2a
(p16INK4a) reverse: GCAGAAGAGCTGCTACGTGAA;
Tnf (TNF) forward: CTATGTCTCAGCCTCTTCTC;
Tnf (TNF) reverse: CATTTGGGAACTTCTCATCC;
Il6 (IL-6) forward: AAGAAATGATGGATGCTACC;
Il6 (IL-6) reverse: GAGTTTCTGTATCTCTCTGAAG;
IL-1α forward: ACGGCTGAGTTTCAGTGAGACC;
IL-1α reverse: CACTCTGGTAGGTGTAAGGTGC;
IL-1β forward: TGGACCTTCCAGGATGAGGACA;
IL-1β reverse: GTTCATCTCGGAGCCTGTAGTG;
Mcp-1 forward: GCTACAAGAGGATCACCAGCAG;
Mcp-1 reverse: GTCTGGACCCATTCCTTCTTGG;
Pai-1 forward: CCTCTTCCACAAGTCTGATGGC;
Pai-1 reverse: GCAGTTCCACAACGTCATACTCG;
Cxcl-1 forward: TCCAGAGCTTGAAGGTGTTGCC;
Cxcl-1 reverse: AACCAAGGGAGCTTCAGGGTCA;
Cxcl-2 forward: CATCCAGAGCTTGAGTGTGACG;
Cxcl-2 reverse: GGCTTCAGGGTCAAGGCAAACT;
COX2 forward: GCGACATACTCAAGCAGGAGCA;
COX2 reverse: AGTGGTAACCGCTCAGGTGTTG;
iNOS forward: GAGACAGGGAAGTCTGAAGCAC;
iNOS reverse: CCAGCAGTAGTTGCTCCTCTTC; p53
forward: CTCTCCCCCGCAAAAGAAAAA; p53
reverse: CGGAACATCTCGAAGCGTTTA; p19
forward: GGAGCTGGTGCATCCTGACGC; p19
reverse: TGGCACCTTGCTTCAGGAGCTC; Mmp3
forward: CTCTGGAACCTGAGACATCACC; Mmp3
reverse: AGGAGTCCTGAGAGATTTGCGC; Mmp12
forward: CACACTTCCCAGGAATCAAGCC; Mmp12
reverse: TTTGGTGACACGACGGAACAGG; Actb (β-
actin) forward: GATGTATGAAGGCTTTGGTC; Actb
(β-actin) reverse: TGTGCACTTTTATTGGTCTC.
Immunofluorescent staining
Primary MEFs were seeded into 8-well chamber slides
and allowed to attach overnight at 20% oxygen. Cells
were then fixed with 4% paraformaldehyde (PFA) for
10 min, permeabilized with 0.3% Triton X-100 in PBS
for 10 min and blocked with 3% BSA in PBST for 1 hr
in room temperature. Primary antibody incubation was
performed at 4°C overnight and secondary antibody
incubation for 1 hr at room temperature. Primary
antibodies used are as follows: anti-p65 (CST #8242)
and anti-NEMO (Santa Cruz sc-8330). Cell nuclei were
counterstained with Vectashield mounting medium with
DAPI. Five images were acquired for each sample at
60x magnification using an Olympus confocal
microscopy.
Senescence-associated β-galactosidase (SA-βgal)
staining in vitro and in vivo
Primary MEFs were seeded into 6-well plates at 3x104
cells per well, allowed to attach overnight and then
treated with either vehicle or KU-55933 at 10 μM for 72
hours. Fresh fat tissues were preserved in ice-cold PBS
prior to staining. MEFs and adipose tissues were then
fixed in 2% formaldehyde and 0.2% glutaraldehyde in
PBS for 10 minutes followed by incubation with SA-
βgal staining solution (MEFs: pH 5.8; Fat: pH 6.0; 40
μM citric acid in sodium phosphate buffer, 5 μM
K4[Fe(CN)6] 3H2O, 5 μM K3[Fe(CN)6], 150 μM sodium
chloride, 2 μM magnesium chloride and 1 mg/ml X-gal
dissolved in N, N-dimethylformamide) for 16-20 hours
in a 37°C incubator without CO2 injector. To quantify,
ten images were acquired randomly using a bright-field
microscopy at 20x maginification. Total number of SA-
βgal+ cells was normalized to the total cell number
(DAPI) to obtain the percentage of SA-βgal+ cells.
Health evaluation
Health assessments were conducted weekly to assess
the age at onset and severity of numerous age-related
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conditions characteristic of Ercc1-/∆ mice, including
dystonia, ataxia, kyphosis, tremor, muscle wasting,
spontaneous activity and coat condition. In addition,
body weight and grip strength were measured. All aging
symptoms were scored on a scale of 0, 0.5 and 1, except
for dystonia on a scale from 0 to 5. The sum of aging
scores was used to determine the overall health of the
individual animals, then averaged by genotype and age
group.
MDSPC isolation
The Atm+/-, Ercc1-/∆Atm+/- and Ercc1-/∆ mice were
sacrificed at 16 to 18 weeks of age, and MDSPC
isolation was performed via a modified preplate
technique as previously described [78, 79]. Briefly, the
skeletal muscle tissue was minced and processed
through a series of enzymatic dissociations: 0.2% of
collagenase type XI (C7657, Sigma-Aldrich) for 1 hour,
2.4 units/ml of dispase (17105-041, Invitrogen) for 45
minutes, and 0.1% of trypsin-EDTA (15400-054,
Invitrogen) for 30 minutes at 37°C. After enzymatic
dissociation, the muscle cells were centrifuged and
resuspended in proliferation medium (Dulbecco’s
modified Eagle’s medium (DMEM, 11995-073,
Invitrogen) supplemented with 10% fetal bovine serum
(FBS, 10437-028, Invitrogen), 10% horse serum (HS,
26050-088, Invitrogen), 0.5% chicken embryo extract
(CEE, CE650T-10, Accurate Chemical Co.), and 1%
penicillin-streptomycin (15140-122, Invitrogen). The
cells were then plated on collagen type I (C9791,
Sigma-Aldrich) coated flasks. Different populations of
muscle-derived cells were isolated based on their
different adhesion characteristics. After 7 days, late
preplate populations (slow-adhering cells), which have
previously been described to contain the MDSPC
fraction of cells, were obtained and cultured in
proliferation medium [80].
MDSPC proliferation assay
MDSPCs were cultured at 5,000 cells per well in
collagen-coated 24-well plates in proliferation medium.
Cell proliferation was tested with an MTS assay. After 3
days, proliferation medium was removed from wells
and 100 µl of fresh proliferation medium was added to
each well and allowed to equilibrate for 1 hr. Then, 20
µl of MTS reagent (Promega, Cat# G3582) was added
to each well and incubated for 4 hrs. The optical density
at 490 nm was measured using a spectrophotometer.
Myogenic differentiation assay and fast myosin
heavy chain staining
The cells were plated on 24 well plates (30,000 cells/well)
in differentiation medium (DMEM supplemented with 2%
FBS). Three days after plating, immunocytochemical
staining for fast myosin heavy chain (MyHCf) was
performed. Cells were fixed for 2 minutes in cold
methanol (-20°C), blocked with 10% donkey serum (017-
000-121, Jackson ImmunoResearch) for 1 hour and then
incubated with a mouse anti-MyHCf (M4276, 1:250;
Sigma-Aldrich) antibody for 2 hours at RT. The primary
antibody was detected with an Alexa 594-conjugated
anti-mouse IgG antibody (A21203, 1:500; Molecular
probes) for 30 minutes. The nuclei were revealed by 4, 6-
diamidino-2- phenylindole (DAPI, D9542, 100ng/ml,
Sigma-Aldrich) staining. The percentage of differentiated
myotubes was quantified as the number of nuclei in
MyHCf positive myotubes relative to the total number of
nuclei.
Histologic analysis
Tissues were fixed in 10% neutral buffered formalin
(NBF) overnight before embedding in paraffin. 5 μm
sections were acquired using a microtome. Hematoxylin
and eosin (H&E) staining was conducted following a
standard protocol.
Glycosaminoglycan (GAG) analysis
Snap frozen lumber spines were harvested at 12 weeks
of age from Ercc1-/Δ and Ercc1-/∆Atm+/- mice. Nucleus
pulposus (NP) tissue was isolated and dissected under a
microscope and six lumbar intervertebral discs were
pooled for analysis. GAG was isolated by papain
digestion at 60°C for 2 hrs. Concentration of GAG was
measured according to the 1,9-dimethymethylene blue
(DMMB) procedure using chondroitin-6-sulfate (Sigma
C-8529) as the standard. DNA concentration was
measured using Pico Green assay (Molecular Probes).
Fold change of GAG content was calculated by
normalizing GAG to DNA concentration.
Micro-computed tomography
Micro-computed tomography (μCT) of spines isolated
from 12-week-old wild type (WT), Ercc1–/Δ and Ercc1–
/ΔAtm+/- mouse littermates were acquired using the
VivaCT 40 scanner (Scanco, Switzerland) with settings:
energy 55 kvP, intensity 145 μA, integration time
200ms, isotropic voxel size 15 μm, threshold 235. 3D
morphometric analysis were performed on lumbar
vertebrae segments (L3-S1).
Statistical analysis
All values were presented as mean+/-S.E.M. Microsoft
Excel and Graphpad Prism 6 were used for statistical
analysis. Two-tailed Student’s t-test was performed to
determine differences between two groups. A value of p
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<0.05 was considered as statistically significant, shown
as *p <0.05, **p <0.01, and ***p < 0.001.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
FUNDING
This work was supported by NIH grants PO1AG043376
(PDR, LJN), R01AG063543 (LJN), R56AG059676
(LJN), R56AG4059675 (PDR), P01AG062412 (PDR),
U19AG056278 (PDR/ LJN) and AG044376 (NV) and a
grant from Glenn/AFAR (LJN). We are also grateful for
the support of The Scripps Research Institute Histology
Core and Animal Research Center at the Florida
campus.
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SUPPLEMENTARY MATERIALS
Supplementary Figures
Supplementary Figure 1. DDR and NF-κB are activated in tissues from progeroid Ercc1-/Δ and old WT mice. Densitometric
analysis of Western Blot shown in Figure 1A (A), Figure 1B (B), Figure 1C. (C) and Figure 1D (D). (A–B) Two-tailed Student’s t-test was performed. (C–D) One-way ANOVA was performed. *p <0.05, **p <0.01 or as indicated in the figure.
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‘
Supplementary Figure 2. Atm haploinsufficiency reduces senescence by regulating NF-κB activation. Densitometric analysis of
Western Blot shown in Figure 7A (A), Figure 7B (B), Figure 7C (C), Figure 7D (D), Figure 7E (E) and Figure 7F (F). P values were determined using a Student’s t-test. *p <0.05, **p <0.01, ***p<0.001 or as indicated in the figure.
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Supplementary Figure 3. Genetic reduction or pharmacologic inhibition of Atm or NF-κB attenuates aging phenotypes and reduces cellular senescence in vivo. (A) qRT-PCR analysis of mRNA expression in liver from 12-week-old WT, Atm+/-, Ercc1-/Δ and Ercc1-
/ΔAtm+/- mice. n=3-6 per group. (B) qRT-PCR analysis of mRNA expression in liver from 10 to 12-week-old WT, p65+/-, Ercc1-/Δ and Ercc1-/Δp65+/- mice. n=4-5 per group. (C) mRNA expression of senescence markers in the kidney of 12-week-old Ercc1-/Δ mice treated with 10 mg/kg of KU-55933 intraperitoneally 3 times per week for two weeks. n = 3 per group. Graphs represent mean+/- s.e.m. P value was determined using Student’s t-test. *p<0.05, **p<0.01, ***p <0.001, ****p <0.0001.